| Objectives: Benign prostatic hyperplasia?BPH?is characterized as a non-malignant enlargement of the prostate,which is one of the most common diseases in ageing men.Surgical intervention is an ideal way for treatment of patients with moderate-to-severe LUTS.Urinary tract infection,urinary frequency,urgency,urodynia and haemorrhage are common post-operative complications of thulium laser resection of the prostate?Tm LRP?.Our study mainly focuses on the role of finasteride in prostate wound healing through AR signalling.In this study,we established Tm LRP beagle models with different treatments.The process of re-epithelialization and changes of inflammatory response are assessed.Besides the in vivo experiment,we also identified the effect of DHT and AR signaling and inflammatory response on prostate epithelial cells in vitro.Materials and methods: 1.A total of 24 healthy adult male beagles aged 2 years were obtained from Shanghai General Hospital.The animals were randomly divided into 3 groups: testosterone group,finasteride group and control group.Animals in testosterone group and finasteride group were fed with 80 mg testosterone undecanoate?Catalent,France,Beinheim S.A.?for 1 week or finasteride?Merck & Co.,Inc.,Kenilworth,NJ,USA?at a dose of 0.5 mg kg-1 body weight per day for 3 months,respectively,before the Tm LRP until they were killed.Serum and intra-prostatic testosterone and DHT level were determined.Histological analysis was conducted to study the re-epithelialization and inflammatory response of the prostatic urethra in each group.2.Prostate epithelial cells overexpressing wild-type androgen receptor were established by lentivirus transfection and the followingpuromycin selection,which termed as BPH-1-AR and Pr EC-AR.MTT assay,Clonogenic assays,fluorescence activated cell sorting?FACS?analysis of cell cycle assay,immunofluorescence assays and western blot analysis were conducted to determine the effect of androgen on the prostate epithelial cells.3.Immunohistochemistry,quantitative real-time PCR analysis and western blot analysis were performed to detect the expression of TNF-? on wound.The distribution of macrophages was detected by CD86 staining of immunofluorescence.For detection of urine TNF-? production,morning urine samples were collected from each canine through the bladder fistula on days 3,5,7,10,14,21,28,and before the thulium laser surgery.Urinary TNF-? quantification was assessed by ELISA commercial kit,which is specific for canine TNF-? evaluation?Ray Biotech,Inc.,Norcross GA,USA?.THP-1 monocytes were infected with particles for sh RNAs targeting AR?Target sequence: NM000044?and then treated with DHT.Conditioned medium was collected from THP-1 culture after the incubations described above.The cytokine?TNF-??in the supernatants was determined using a commercial ELISA kit?R&D Diagnostics,MN,USA?.4.The expression of TNFR1 in prostate epithelial cells was knocked down then we treated these cells with differect dose of TNF-?.The proliferation of prostate epithelial cells was tested by MTT assays.Western blot analysis was performed to detect the expression of cell cycle related factors affected by TNF-?.Fluorescence activated cell sorting analysis of cell apoptosis assay were conducted to explore the effect of TNF-? on apoptosis.We further investigated the effect of TNF-? on prostate fibroblasts.Wound healing and assay of collagen gel contraction were performed.Results: 1.Testosterone and DHT level increased in testosterone group and DHT decreased in finasteride group.Accelerated wound healing of prostatic urethra was observed in the finasteride group.Epithelium in both groups was much thicker than that in testosterone group.Faster than testosterone group,re-epithelialization was completed in finasteride group and control group 4 weeks after surgery.2.Prostate epithelial cells overexpressingwild-type androgen receptor were established by lentivirus transfection and the following puromycin selection,which termed as BPH-1-AR and Pr EC-AR.DHT significantly reduced number of colonies and cell viabilities through a concentration-dependent way.Ki67 staining also indicated that the proliferation of BPH-1-AR and Pr EC-AR was remarkably reduced by DHT.Androgen-dependent AR-mediated growth suppression was associated with increased expression of the CDK inhibitors p21,instead of p27,and subsequently reduced expression of cyclin D1.Flow cytometric analyses of DNA content documented that DHT exposure of prostate cells induced cell growth arrest in phase G0/G1.3.We stained M1 macrophages with CD 86+ primary antibodies and found that M1 macrophages were significantly reduced in finasteride and control group,especially in first and second week.Immunohistochemistry analysis manifested that TNF-? protein level was increased in testosterone group when compared with finasteride and control group in 1-2 weeks.Similarly,real-time PCR and western blot analysis also revealed that the expression of TNF-? increased in the wounds of the testosterone group in 1-3 weeks.At the same time,TNF-? expression was reduced in finasteride group when compared with control group,especially in first and second week.In addition,we found that TNF-? was remarkably reduced in urine,especially in the early stage?day 3 to day 5?in finasteride group,whereas testosterone treatment immensely enhanced and prolonged TNF-? creation.4.Lower level of TNF-??1 ng m L-1?had no effect on proliferation of prostate epithelial cells,whereas TNF-? at higher level?50 ng m L-1?inhibited cell growth through reducing the expression of ?-catenin and cyclin D1 we also explored the role of TNF-? on apoptosis.No notable apoptosis of Pr EC was induced by 50 ng m L-1 TNF-?,which is in line with the previous study.Wound closure model in the presence or absence of 20 ng m L-1 TNF-? showed that TNF-? prevented wounds closure,but TNF-? showed loss of the inhibitory effect on wound closure of cells lack TNFR1.We found TGF-? could also increase the ?-SMA expression inprostate fibroblast cells?WPMY-AR and Pr SC-AR?.However,treated with both TGF-??1 ng m L-1?and TNF-??5 ng m L-1?could reverse the ability of TGF-? to up-regulate ?-SMA,indicating that TNF-? has antagonistic activity against TGF-? in prostate fibroblasts.TNF-? treatment leads to phosphorylation of JNK,and blockade of JNK pathway by SP600125?30 m M?reversed suppression effect of TNF-? on ?-SMA.Collagen gel contraction mediated by WPMY-AR and Pr SC-AR was inhibited by 68.4% and 48.1% when treated with TNF-?.Wound-healing assays also revealed the inhibition role of TNF-? on prostate fibroblasts.Conclusion: Testosterone treatment repressed re-epithelialization and wound healing of prostatic urethra.Finasteride treatment may be an effective way to promote prostate re-epithelialization... |
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